Apoptosis-inducing small molecules typically engage the intrinsic pathway, in which release of mitochondrial cytochrome c induces activation of caspase-9, followed by activation of caspase-3. The rate of apoptosis is dependent on the type and strength of the apoptotic stimuli and the cell type, and a minimum of several hours of activating stimulus is typically required for apoptosis induction through the intrinsic pathway (Goldstein et al., 2000). This lengthy induction period is likely due to rate limiting steps upstream of cytochrome c release such as transcription/translation (Dudgeon et al., 2009; Fridman and Lowe, 2003) or cell cycle dependent responses (Hamada et al., 2009). While the time to cytochrome c release varies, once initiated, cytochrome c release may be complete within 5-10 minutes (Goldstein et al., 2005; Luetjens et al., 2001) regardless of cell type. The kinetics of caspase activation following cytochrome c release may be dependent on the cell type, although in certain cells caspase activation is complete within 20 minutes after initiation (Luo et al., 2001; Rehm et al., 2002).
Agents capable of inducing intrinsic pathway-mediated apoptosis are widely employed in a range of biochemical experiments. Typically, the broad-spectrum kinase inhibitor staurosporine has been the small molecule of choice, as it requires a short time period for induction of apoptosis relative to other agents. Among numerous other experiments, staurosporine has been employed in studies that have identified fundamental regulators of the apoptotic pathway (including Bcl-2 (Yang et al., 1997), CAD/ICAD (Sakahira et al., 1998), AIF (Susin et al., 1999), and multiple others), in proteomics experiments examining the scope of cellular caspase protein substrates (Agard et al., 2012; Dix et al., 2008; Dix et al., 2012; Shimbo et al., 2012), and to help elucidate mechanisms of apoptotic death as induced by small molecules (Wolpaw et al., 2011). However, even staurosporine requires multiple hours for full cytochrome c release from the mitochondria (Bossy-Wetzel et al., 1998; Botham et al., 2014), with apoptotic cell death ensuing. In addition, the pan-kinase inhibition and ever-emerging biological effects elicited by staurosporine (Savitski et al., 2014) complicate interpretation of downstream readouts, as demonstrated by the fact that other proapoptotic agents (e.g. doxorubicin and bortezomib) induce a different pattern of caspase cleavage from staurosporine (Shimbo et al., 2012). A compound that rapidly induces mitochondrial cytochrome c release and apoptosis without prolonged engagement of upstream processes would be especially valuable in these and other cell biology experiments.
The overarching impact of apoptosis in diseases, such as cancer (Fulda, 2007), heart disease (Narula et al., 2006), and neurodegeneration (Ferrer, 2006), highlights the necessity of having resources to study and find regulators involved in programmed cell death.